In certain disease states, the genetic component predominates over environmental factors. These diseases are termed genetic disorders and typically fall into one of three categories: (1) disorders characterized by the absence, excess, or abnormal arrangement of one or more chromosomes; (2) Mendelian or simply-inherited disorders, primarily caused by a single mutant gene and subclassified into autosomal dominant, autosomal recessive, or X-linked types; and (3) multifactorial disorders caused by interaction of multiple genes and environmental factors. The economic cost of genetic disorders is enormous. For example, the annual U.S. expenditure on treatment for the genetic disease cystic fibrosis alone is estimated at about $500 million. Cystic fibrosis and other genetic diseases account for the presence of at least one-third of the children in pediatric hospitals. Thompson & Thompson, Genetics in Medicine (4th ed. 1986). The psychological and emotional costs to those afflicted with genetic disorders and their families are incalculable.
Prenatal diagnosis and genetic counselling are important in detecting, preventing, and treating genetic disorders. If no abnormality is detected, parents' worries are diminished as the fetus is carried to term. If a fetal abnormality is detected sufficiently early, parents have the option of aborting the pregnancy. Occasionally, antenatal detection of an abnormality may allow its treatment before birth, or facilitate its treatment immediately after birth by notifying doctors when treatment is required and allowing for early preparation.
Techniques available for performing prenatal diagnosis include maternal serum screening, ultrasonography, chorionic villus sampling, fetal tissue screening, and amniocentesis. See, e.g., Kingston, Brit. Med. J. 298:1368-71 (1989); Platt & Carlson, N. Engl. J. Med. 327:636-638 (1992); The Unborn Patient--Prenatal Diagnosis and Treatment (Harrison, et al. eds., W. B. Sanders, Philadelphia, Pa. 1991) (each of which is hereby incorporated by reference in its entirety for all purposes). The techniques differ in their timing, safety, accuracy, rapidity, and scope of diagnostic markers they can detect. Early diagnosis is advantageous in facilitating therapeutic treatment, reducing the risk to the mother should an abortion be necessary, and in relieving parental anxiety should no problem be found.
Maternal serum screening involves virtually no risk to the fetus, but is useful only for detecting a few abnormalities such as neural tube defects. Ultrasonography also is safe, but can detect only structural characteristics, such as fetal size. Biochemical or genetic abnormalities cannot be detected using this method. Ultrasonography is typically used to determine fetal age and position in the womb in preparation of performing other diagnostic techniques. See, e.g., Hoybe in The Unborn Patient--Prenatal Diagnosis and Treatment (Harrison, et al. eds., W. B. Sanders, Philadelphia, Pa. 1991), ch. 8.
Chorionic villus sampling is a recently developed alternative to conventional amniocentesis. Chorionic villi are protrusions in the outermost extraembryonic membrane surrounding the fetus. In this method, chorionic villus tissue may be biopsied from 8 weeks gestation onward. Fibroblasts in the sample are cultured and subjected to biochemical or genetic testing. Typically 3-4 weeks are required to obtain a sufficient cell number to perform tests reliably. The early time at which chorionic villus sampling can be performed, as compared with conventional amniocentesis, is advantageous for the reasons discussed above. However, the increased risk of miscarriage or fetal deformation following chorionic villus sampling is four times greater than that following a conventional amniocentesis. See, Kingston, supra.
Fetoscopy involves obtaining tissue directly from the fetus and is performed during the second trimester of pregnancy. Cells can be cultured from the fetal tissue and subjected to biochemical and genetic tests. Fetoscopy involves five-fold higher risk to the fetus than amniocentesis. Thompson & Thompson, supra).
Embryo biopsy is possible at the 8-16 cell stage. See Handyside, N. Engl. J. Med. 327:905-9 (1992). One or two cells are scraped from the embryo, and subject to biochemical or genetic testing. Embryo biopsy is limited to those few pregnancies in which conception is by in vitro fertilization. In vitro fertilization is expensive to perform (see, e.g., San Francisco Examiner (Sep. 24, 1992)) and has a low probability of a successful pregnancy compared with conventional conception.
Amniocentesis (literally, tapping of the amnion) refers to a procedure of removing a sample of amniotic fluid transabdominally with a syringe. Conventionally, amniocentesis involves extraction of about 20 ml amniotic fluid from the amnionic sac at 14-16 weeks' gestation, when such volumes become available. Amniotic fluid contains mainly cells shed from the skin and digestive and urinary tracts of the fetus. Some diagnostic tests for proteins, such as .alpha.-fetoprotein, can be performed directly on the fluid. However, most tests require culturing cells within the fluid in order to provide sufficient cells or DNA for reliable analysis. In conventional amniocentesis, the cultured cells are fibroblasts and 3-4 weeks are required to achieve a sufficient cell number and density to perform diagnostic tests. Mandon & Mathieu, J. Inher. Metab. Dis. 12 suppl. 2:257-59 (1989). Extraction of the fluid causes an increased risk of miscarriage of about 0.5%. Thompson & Thompson, supra.
There have been several recent reports discussing amniocentesis performed earlier than the usual time of 14-16 weeks. In these reports, the volume of amniotic fluid extracted is generally smaller than in conventional amniocentesis, but the method of culturing cells in the fluid is the same. Elejalde, et al. report that amniocentesis is possible from the ninth week of pregnancy. Am. J. Med. Gen. 35:188-196 (1990). However, they found that early amniocentesis causes higher rates of fetal losses and amniotic fluid leakage. Elejalde, et al. also report that a longer time was required to obtain test data following early amniocentesis, because a smaller volume of fluid and, hence, fewer cells could be obtained and, therefore, longer culture periods were required.
Nevin, et al. also report that amniocentesis is possible from the ninth week of pregnancy. Prenatal Diagnosis 10:79-83 (1990). In this relatively small-scale experiment (222 patients), these authors found no increased risk of fetal loss compared with conventional amniocentesis. However, Nevin, et al. were able to extract only a much smaller volume of fluid (e.g., 4 ml for amniocentesis at 9 weeks) than the typical 20-ml volume extracted at 14-16 weeks.
Evans, et al. discuss early amniocentesis from 10-14 weeks of pregnancy. Am. J. Obstet. Gynecol. 160: 1332-9(1989). Evans, et al. report more frequent failure of cell cultures derived from early amniocentesis because of the difficulties associated with the smaller volume of fluid that could safely be extracted.
Holzgreve and Miny identify several problems when amniocentesis is performed earlier then 15 weeks. Seminars in Perinatology 13:260-277 (1988). These authors discuss the risks of withdrawing a greater proportion of total amniotic fluid than is the case for conventional amniocentesis performed at 14-16 weeks. These authors also discuss laceration of the extraembryonic celoma space or yolk sac and difficulties in interpreting a positive acetylcholinesterase test in early amniocentesis.
From the preceding discussion, it is apparent that withdrawing smaller volumes of amniotic fluid earlier in pregnancy and culturing cells by conventional methods is not an ideal means of performing prenatal diagnosis. Moreover, although alternative methods to amniocentesis are available, they lack the safety or general applicability of amniocentesis. Therefore, a need exists for improved methods of prenatal diagnosis that can be performed earlier in pregnancy and/or with greater safety, and/or rapidity, and/or accuracy than conventional amniocentesis. The present invention exploits a new method of culturing cells in amniotic fluid to fulfill this and other needs.